A change in the state of cells or response of cells to a chemical or the like has conventionally been observed as if the average of a cell group represented the property of one cell. In an actual cell group, cells which are synchronized in a cell cycle are not so many and cells express a protein at cycles different from each other. A method of synchronized culture has been developed with a view to overcoming these problems. Since the cells cultured are not cells derived from exactly the same cells, however, there is a possibility of causing a difference in the protein expression due to a difference in genes among the derived cells before cultivation. Upon actual analysis of a response to stimulation, it is very difficult to find whether fluctuations appearing in the results are attributable to the fluctuations in response which are common in the reaction mechanism of the cell itself or to a difference among cells (that is, a difference in genetic information). Since a cell line is usually not obtained by cultivating one cell, it is also very difficult to find whether or not the fluctuations in the reproduction of a response to stimulation are attributable to a genetic difference among cells. Stimulation (signal) to cells can be classified into two groups, that given by the amounts of a signal substance, nutrition and dissolved gas contained in a solution surrounding the cells therewith; and that given by a physical contact with another cell, which also makes the judgment on the fluctuations difficult.
When cell observation is carried out in the biotechnological research field, it is common practice to temporarily take out a portion of a cell group cultured using a large-sized incubator and observe the cells set on a microscope, or to control the temperature of the microscope while surrounding the entire microscope with a plastic container and carry out microscopic observation at a carbon dioxide concentration and humidity controlled using another small container inserted in the plastic container. Upon this observation, the conditions of a culture solution are kept constant by replacing a wasted culture solution with a fresh one, while culturing cells. According to the method as disclosed in Japanese Patent Laid-Open No. 10-191961, for example, the nutrition condition is kept constant by a mechanism in which a circulation pump moves the level of a medium relative to the surface of a base material up and down between a level higher than the upper end height of the base material and a level lower than its bottom end height, whereby a new medium is fed when the level is below the above-described low level and the medium is discharged when the level exceeds the high level. Disclosed in Japanese Patent Laid-Open No. 08-172956 is a culture apparatus, in which one end of each of an inlet pipe for introducing a new culture medium into a culture vessel, an outlet pipe for discharging the culture medium in the culture vessel to the outside, and a gas pipe permitting a gaseous portion in the culture vessel and a pump to communicate with each other is inserted in the culture vessel and filters for preventing the invasion of bacteria into the culture vessel are installed to the inlet pipe, outlet pipe and gas pipe, respectively. Thus, the nutrition conditions in a culture tank can be kept constant. In either one of these inventions, however, an example of culturing cells while controlling their solution environment and physical contact between cells is not known.
With a view to overcoming these problems, the inventors of this application invented a technology of selecting only one specific cell and cultivating it as a cell line, a technology of controlling the conditions of a solution environment of cells and keeping the cell density in the vessel constant upon cell observation, and a technology of cultivating and observing cells, which interact with each other, while specifying them and applying for patent on them as Japanese Patent Application No. 2000-356827.
The microchamber newly proposed by the present inventors has novel characteristics in its constitution. The microchamber is formed utilizing a microfabrication technology of glass or the like. Prior to the initiation of cultivation, the microchamber is formed on the glass surface and cell culture can be carried out by making use of its shape. It is therefore difficult to change, depending on the state after cultivation, the pattern of a flow path between the microchambers which path determines interaction between the microchambers. It is also difficult to change the shape of the microchamber itself in accordance with the progress of the cultivation.
In addition, a technology of heating the microchamber to change its shape by making use of a focused beam or the like cannot be applied to the heating in a three-dimensional local region smaller than the wavelength of infrared light.
An object of the present invention is therefore to provide a novel microchamber whose shape can be changed depending on the cultivation stage, based on the detailed investigation on the above-described microchamber developed by the present inventors. Another object of the present invention is to provide a novel microchamber for cell culture which permits spot heating of a nanoscale fine region.